Oral Presentation 25th International Pathogenic Neisseria Conference 2026

The Compensatory Mutation with homology to the mleN malate-lactate antiporter that increases the fitness of antibiotic resistant Neisseria gonorrhoeae is a lysine transporter (139509)

Stephanie L Bishop 1 , Kate Newns 2 , Maria X Cardenas Alvarez 2 , Julia Adug 3 , Ian A Lewis 3 , Yonatan H Grad 4 , Ann E Jerse 5 , Robert A Nicholas 2
  1. Biochemistry, Western University , London, Ontario, Canada
  2. University of North Carolina at Chapel Hill, Chapel Hill, NC, United States
  3. Biological Sciences, University of Calgary, Calgary, Alberta, Canada
  4. TC Chan School of Public Health, Harvard University, Boston, Massachusetts, USA
  5. Microbiology & Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA

The rise in antibiotic-resistant strains of Neisseria gonorrhoeae (Ng) threatens the continued use of antibiotics to treat gonococcal infections. Because resistance occurs primarily through mutation of the essential proteins targeted by the antibiotics, the resulting mutated targets often confer a fitness cost of reduced growth and pathogenic potential, and thus resistant strains are thought to acquire additional compensatory mutations that increase fitness. To identify potential compensatory mutations, we carried out co-infections of FA19 and FA19 harboring the penA gene from the ceftriaxone-resistant strain H041 (penA41) in the female mouse model and isolated several mutants that conferred a robust fitness advantage to the resistant strain. One of these compensatory mutations was a codon deletion (ΔA467) in a gene with 27% identity to Bacillus subtilis MleN, previously shown to be a Na+-dependent malate-lactate exchanger, but the substrates exchanged by Ng MleN were unknown. Growth analyses in Graver-Wade medium revealed that both FA19 and FA19 penA41 strains harboring the mleNΔA467and mleN:kan (KO) genes exhibited a delayed growth profile compared to those strains with the mleNWTgene. Semi-targeted extracellular metabolomics profiling showed that all strains utilized lactate as the primary carbon source and switched to glucose and amino acid consumption upon lactate depletion. Moreover, mleN mutant and KO strains did not consume lysine and displayed decreased excretion of TCA cycle intermediates malate, succinate, and fumarate during growth compared to mleNWT parent strains. Removing lysine from the growth medium decreased the exponential-phase growth rate and the excretion of TCA cycle intermediates of mleNWT strains such that all the strains grew at the same rate and exhibited similar dicarboxylic acid metabolic profiles. These data suggested that MleN functions as a lysine transporter. To test this hypothesis, we generated a construct that disrupts the lysA gene that converts m-diaminopimelic acid to lysine, the last step in de novolysine biosynthesis. Transformation experiments demonstrated that disruption of lysA could be achieved in strains with mleNWT (endogenous or complemented) but not in strains with mleNΔA467 or mleN:kan. These data demonstrate that MleN is responsible for lysine import in Ng.